Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Microorganisms capable of degrading neonicotinoids and their metabolic pathways: a review / 生物工程学报

Neonicotinoid compounds are usually considered harmless and eco-friendly in terms of their targeted toxicity compared to that of pyrethroids and phosphorus-containing pesticides. However, overuse of neonicotinoid insecticides resulted in the accumulation of its residuals or intermediates in soil and water, which consequently affected beneficial insects as well as mammals, yielding pollution and secondary risks. This review summarized the recent advances in neonicotinoid degrading microorganisms and their metabolic diversity, with the aim to address the urgent need for degrading these insecticides. These advances may facilitate the development of controllable and reliable technologies for efficiently transforming neonicotinoid insecticides into value-added products by synthetic biology and metagenomics.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Carbaryl degradation by bacterial isolates from a soil ecosystem of the Gaza Strip

Abstract Carbaryl is an important and widely used insecticide that pollutes soil and water systems. Bacteria from the local soil ecosystem of the Gaza Strip capable of utilizing carbaryl as the sole source of carbon and nitrogen were isolated and identified as belonging to Bacillus, Morganella, Pseudomonas, Aeromonas and Corynebacterium genera. Carbaryl biodegradation by Bacillus, Morganella and Corynebacterium isolates was analyzed in minimal liquid media supplemented with carbaryl as the only source of carbon and nitrogen. Bacillus and Morganella exhibited 94.6% and 87.3% carbaryl degradation, respectively, while Corynebacterium showed only moderate carbaryl degradation at 48.8%. These results indicate that bacterial isolates from a local soil ecosystem in the Gaza Strip are able to degrade carbaryl and can be used to decrease the risk of environmental contamination by this insecticide.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Efficacy of Spodoptera litura multiple nucleopolyhedrovirus after serial passage through the homologous insect larval host.

An originally isolated baculovirus, Spodoptera litura multiple nucleopolyhedrovirus (SpltMNPV) was serially passed through the S. litura larvae for upto four generations to determine the mean number of occlusion bodies (OBs) harvested per larva and their efficacy in terms of infectivity, feeding cessation and speed of kill of host larvae. The results revealed that the mean number of OBs harvested per larva increased significantly with increase in the dose of SpltMNPV at each passage and the yield was significantly lower in original stock wild-type SpltMNPV (P0) as compared to serially passed SpltMNPV (P1, P2, P3 and P4). Laboratory bioassays indicate that median lethal doses (LD50), median times to feeding cessation (FT50) and median survival times (ST50) of P0, P1, P2, P3 and P4 were significantly different from each other. The OBs of each passage when tested for their cross-infectivity against Spodoptera exigua and Spilarctia obliqua revealed significant reduction in their mortality. These results indicate that serially passed SpltMNPV is more host specific and more effective biocontrol agent than the original stock wild-type virus and can be adopted for mass production as a viral pesticide for control of the S. litura.

Influence of endosulfan and monocrotophos exposure on the activity of NADPH cytochrome C reductase (NCCR) of Labeo rohita (Ham).

The response of NADPH cytochrome C reductase (NCCR) activity in liver of Labeo rohita fish exposed to the pesticides, 0.25 microgl(-1) endosulfan and 2 mg/l monocrotophos was studied. In terms of specific enzyme activity (mU/mg protein) a significant level of NCCR was observed in the liver tissues of Labeo rohita exposed to the pesticides, when compared to the control fish (2.460 mU/mg protein). Increase of NCCR activity was more in the liver of the fish exposed to monocrotophos (4.595 mU/mg protein) than those exposed to endosulfan (2.850 mU/mg protein). The results demonstrate that the pesticides, endosulfan and monocrotophos, interfere with NADPH dependent monoxygenase mechanism and are effective inducers of NADPH cytochrome C reductase. The activity of NCCR in the liver tissue of Labeo rohita may serve as a useful tool for monitoring aquatic pollution.

Devicyprin induced gonadal impairment in a freshwater food fish, Channa punctatus (Bloch).

Synthetic pyrethroids are the recent major class of broad spectrum, photostable, organic insecticides used in agricultural, domestic and veterinary applications and now account for more than 30% of global insecticide use. Cypermethrin is metabolized and eliminated significantly more slowly by fish than by mammals or birds, which may explain this compound's high toxicity in fish compared to other organisms. The present communication deals with histoanatomical alterations in the gonads of a local fresh water food fish, Channa punctatus exposed to 0.033 ppm (96 hr LC50 X 1/10) concentration of a synthetic pyrethroid, devicyprin (cypermethrin 25%) in aquatic medium of aged tap water for 15, 30 and 45 days respectively. In testis, exposure dependent histological damage has been observed in terms of vacuolization, condensation of spermatogonic cells, distortion of tubular epithelium, shrinkage of interstitial cells and general inflammatory responses. Longest exposure of 45 days has resulted in peculiar starry-sky appearance of the testicular tissue. Gross histo-anatomy of ovarian tissue reveals epithelial lesions, inflammatory responses, stromal hemorrhage, increased interstitium and shrinkage of yolk vesicles towards periphery These findings are quite suggestive of reproductive impairments leading to delayed gonadal maturity and adversely affecting processes of sperm production and ovulation and thus, the fish production.

Resíduos de tiametoxam, aldicarbe e de seus metabólitos em folhas de cafeeiro e efeito no controle de Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae) / Residues of thiamethoxam, aldicarb and its metabolites in coffee leaves and effect on the Control of Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

O bicho-mineiro Leucoptera coffeella (Guérin-Mèneville), uma das pragas mais importantes da cafeicultura brasileira, é controlado principalmente com inseticidas. O objetivo deste trabalho foi estudar os resíduos e a translocação do inseticida tiametoxam em folhas de cafeeiros, bem como avaliar seu efeito no controle do bicho-mineiro, comparando-o com o aldicarbe, utilizado como padrão. Para isto, foi instalado um experimento no município de Garça, SP, no período de dezembro/2001 a agosto/2002. Os tratamentos utilizados foram: aldicarbe 150 G, nas doses de 2,25 e 4,50 g i.a./cova, tiametoxam 10 GR, nas doses de 0,15 e 0,30 g i.a./cova e testemunha (sem aplicação). Amostras de ramos foram colhidas em pré-contagem e aos 30, 60, 90, 120, 150, 180, 210 e 240 dias após a aplicação, em três alturas dos cafeeiros (terços inferior, médio e superior), avaliando-se a porcentagem de folhas minadas. As determinações de aldicarbe e seus metabólitos ativos, aldicarbe sulfoxido e sulfona, e os de tiametoxam foram feitas por cromatografia em fase gasosa usando-se detector de nitrogênio-fósforo e de espectrometria de massas, respectivamente. Os resultados indicaram translocação uniforme de ambos inseticidas nos três terços das plantas de café, quando aplicados no solo. Foi constatada também, a maior persistência do tiametoxam, cujos resíduos foram encontrados até oito meses após a aplicação, enquanto os metabólitos sulfóxido e sulfona foram encontrados entre quatro e seis meses após a aplicação. Foi observado controle do bicho-mineiro pela aplicação de ambos inseticidas.

Hydrolysis of organophosphorus compounds by an esterase isozyme from insecticide resistant pest Helicoverpa armigera.

Esterase activity of resistant and susceptible H. armigera were compared in gels with different substrate such as naphthyl acetate, naphthyl phosphate, paraoxon and monocrotophos. Whole body extract of resistant H. armigera hydrolyzed paraoxon, monocrotophos and naphthyl phosphate in gels. Resistant H. armigera showed high esterase, phosphatase and paraoxon hydrolase activity compared to susceptible ones.

Localisation of identical organophosphorus pesticide degrading (opd) genes on genetically dissimilar indigenous plasmids of soil bacteria: PCR amplification, cloning and sequencing of opd gene from Flavobacterium balustinum.

Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18. Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions. The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.

Serotonin and benzylamine oxidation by type A and type B MAO of rat brain in presence of organophosphate pesticides.

Organophosphate (OP) pesticides, monocrotophos (MCP), dichlorvos (DDVP) and phosphamidon significantly inhibit both MAO-A and MAO-B activities in rat brain mitochondria. The inhibition of MAO-A by MCP is reversible whereas the inhibition by DDVP and phosphamidon is irreversible. MAO-B is inhibited irreversibly by all these organophosphates suggesting that the mechanism of action of OP pesticides is through phosphorylation of serine residue present in active centre of MAO.

Subacute toxicity of fenvalerate in broiler chicks: concentration, cytotoxicity and biochemical profiles.

Subacute toxicity study of fenvalerate was carried out in broiler chicks after oral administration @ 525.6 mg/kg once daily for 28 days. The blood concentration of fenvalerate following 1 day post-administration (pd) was 39.65 +/- 2.67 micrograms/ml and maintained plateau thereafter up to day 21 pd, and then declined (18.46 +/- 1.47 micrograms/ml) on day 28 pd. Intestine contained maximum residue (7.46 +/- 1.96 micrograms/g) followed by fat (5.95 +/- 1.16 micrograms/g), brain (5.06 +/- 0.96 micrograms/g), liver (3.93 +/- 0.51 micrograms/g), kidney (3.79 +/- 0.72 micrograms/g) and heart (1.72 +/- 0.35 micrograms/g). Histopathological examinations showed focal areas of necrosis in liver, proliferation and fibrosis of bile duct, larger size of glomeruli, glomerular and tubular necrosis in treated birds. Fenvalerate significantly increased the cholesterol level in brain, GPT activity in liver and heart, GOT activity in heart, and alkaline phosphatase activity in heart and brain tissue. It significantly decreased the glycogen content in liver and heart, GOT activity in brain and acid phosphatase activity in all the tissues analyzed. It appears that comparatively fowl is resistant to fenvalerate toxicity.

Insecticide interaction with carrier and neuroproteins.

Binding of alpha-, beta-, gamma-hexachlorocyclohexane, p,p'-DDT and p,p'-DDE with bovine serum albumin (BSA) and locust brain homogenate was studied. Binding affinities of pesticides were higher for the locust brain homogenates than for BSA. Results of uptake by isolated locust brain revealed higher uptake of gamma-HCH than alpha-HCH. gamma-HCH uptake was also higher from locust haemolymph than either from BSA or from buffer.

Inhibition and recovery of selected target enzyme activities in tissues of penaeid prawn, Metapenaeus monoceros (Fabricius), exposed to different insecticides.

The target enzymes, acetylcholinesterase (for phosphamidon and carbaryl) and Mg2+ ATPase (for DDT and fenvalerate) have been assayed during exposure and reclamation of these insecticides in M. monoceros. Toxicity of these insecticides are in the order: fenvalerate greater than DDT greater than carbaryl greater than phosphamidon. Reclamation studies show that fenvalerate is rapidly degradable while DDT is slowly degradable. It is suggested that pyrethroid, organophosphate and carbamate insecticides may be preferred over organochlorine compounds in farm operations.

Involvement of mono-oxygenases as a major mechanism of deltamethrin-resistance in larvae of three species of mosquitoes.

Role of mono-oxygenases as a mechanism of resistance to the synthetic pyrethroid, deltamethrin in the larvae of Culex quinquefasciatus Say, Aedes aegypti L. and Anopheles stephensi Liston developed by laboratory selections with deltamethrin, DDT or deltamethrin and the synergist, piperonyl butoxide (PBO) in the ratio of 1:5, was investigated. There was a significant correlation with mono-oxygenase activity and larval LC50 to deltamethrin in various strains of all the three species. In addition, the activity of glucose-6-phosphate dehydrogenase (G6PD), the main NADPH generating enzyme for mono-oxygenases, also showed enhanced activity in deltamethrin and DDT-selected strains. The present data, therefore, clearly suggest that deltamethrin resistance in the larvae of Cx. quinquefasciatus, Ae. aegypti and An. stephensi is mainly due to the detoxification of deltamethrin by microsomal mono-oxygenases. High activity of G6PD observed in DDT-selected strains seems to be related to its role as a rate-limiting enzyme in GSH-dependent dehydrochlorination of DDT.